Note: Descriptions are shown in the official language in which they were submitted.
METHOD FOR PRODUCING EPOXIDES
~ND GLYCOLS FROM ALKENES
This invention relates generally to an enzy-
matic process for making useful commercial products from
alkenes. More particularly, the invention relates to an
improved process for the production of epoxides and gly-
cols from alkenes wherein an enzyme is used to produce
an intermediate halohydrin and the halohydrin is conver-
ted to an epoxide or glycol by an enzymatic or chemical
process.
As used herein, the term "alkene~' is intended
to include any open chain hydrocarbon having carbon to
carbon double bonds, wherein each of the carbons con-
taining at least one of the double bonds is joined to
either hydrogen or another carbon. Alkenes include com-
pounds having more than one double bond. The term "ole-
fin'l as used herein is intended to have the same meaning
as "alkene".
A number of useful chemical products, e.g.
surfactants, humectan~s, polymers and plasticizers, are
produced from alkenes such as ethylene and propylene.
Examples of such useful products prepared from alkenes
include epoxides and glycols. The preparation of other
products often involves first producing an epoxide or
2~ glycol and then reacting it in some subse~uent chemical
process to form the desired compound. To form the
epoxide two general approaches have been used
~k
~6~(~3
traditionally. The first of these is to oxidize the
alkene directly. The second is to form an intermediate
halohydrin and then convert it with a base to form the
epoxide. Glycols usually are produced by hydrating the
corresponding epoxide. In recent years the development
of alternate processes for producing epoxides and gly-
cols has been receiving considerable attention, due to
the high commercial value of such materials, the exten-
sive energy requirements of the known processes and the
high cost of required reaction materials.
Processes utilizing direct oxidation, current-
ly a commercially favored path from ethylene to ethylene
oxide, normally require pure oxygen and supported silver
oxide catalyst, both of which add to cost and necessi-
tate expensive safety procedures and devices. Moreover,the elevated temperature required in the process, typi-
cally 270C, increases the energy cost in the process.
Also, reaction yields are decreased by the production of
substantial quantities of carbon dioxide and water as by
products.
Processes involving the production of epoxides
by conversion of an intermediate halohydrin, currently
a commercially favored path from propylene to propylene
oxide, have encountered problems in the formation of the
halohydrin itself. Known processes for the production
of halohydrins from alkenes typically involve the addi-
tion of alkene, halogen and water in a reactor under con-
trolled conditions~ Such processes frequently result in
the production of undesirable side products such as hy-
drochloric acid (which requires neutralization), halo-
alkanes and bis- (haloalkyl) ethers. Moreover, use of
free halogen in any process requires expensive control
procedures and equipment to prevent loss of this tGXiC
reactant. Also, the use of free halogen is now prefer-
ably avoided because of the energy-intensive process em-
ployed for its production.
~6~
It is a principal object of the present inven-
tion to provide an improved process for producing
epo~ides and glycols from alkenes.
Another object of the invention is to provide
a process for producing epoxides and glycols from al~
~enes wherein an intermediate halohydrin is produced
without requiring the use of free halogen.
A further ob~ect of the invention i5 to pro-
vide a process for the production of epoxides and gly-
cols from alkenes which is relatively safe, low in cost,and low in energy requirements as compared with known
processes.
It is a still further object of the present in~
vention to provide a general process for the conversion
of a large number of different alkenes to the epoxide
or glycol through a halohydrin intermediate under con-
ditions wherein the necessity for halohydrin isolation
is avoided.
Other objects of the present invention will
~0 become more apparent from the following detailed descrip-
tion and accompanying claims. II1 the description and
claims, all proportions and percentages are by weight,
all pressures are stanaard atmospheric, and all tempera-
tures are in degrees centigrade, unless otherwise speci-
fied.
In general, in accordance with one aspect ofthe present invention, the halohydrin of an olefin is
produced by introducing the olefin into a reaction mix-
ture of a halogenating enzyme, a source of halide ion,
and an oxidizing agent. The reaction of the olefin to
pxovide the halohydrin of the olefin proceeds spontane-
ously and rapidly under ambient conditions of tempera-
ture and pressure. The halohydrin is then converted to
the epoxide or glycol by an enzymatic or chemical pro-
cess.
As used herein, the term "epoxide" includes
- ~ -
oxides and the term "glycol" is the same as diol where-
in the hydroxyl groups are on adjacent carbons.
The enzymatic halogenating process of the
present invention has several advantages over the pres-
ent state of the art for producing halohydrins fromolefins, including the following: The use of inexpen-
sive, less dangerous, inorganic halide, rather than
elemental halogen, i.e. bromide ion rather than bromine;
use of ambient temperature; and use of standard or close
to standard atmospheric pressure.
In addition to proceeding favorably at room
temperature, this enzymatic process involves the use of
dilute H2O~, not necessari]y purified. The H2O2 may be
added directly or generated in situ by an enzymatic or
chemical reaction. ~his reduces the cost of the H2O2
as compared to the cost of concentrated, purified materi-
al; increases the safe usage of the substance; and ex-
tends the life of the halogenating enzyme. The in situ
generation of peroxide will be discussed below.
The olefins useful in the process can be
broadly defined as any hydrocarbon containing a carbon
to carhon double bond, represented by the following
structural formula:
R~ R3
2 C = C 4
wherein each o~ Rl, R2, R3 and R4 is selected from a
group consisting of:
(1) hydrogen
~2) a straight chain )
saturated or unsaturated
(3) a branched chain ) hydrocar~on radical havi~g
~4) a cyclic ) from 1 to 12 carbon atoms;
and provided that all direct linkages to the carbons of
the double bond are either hydrogen or carbon, that
none of the R groups are themselves`connected such as to
form a cyclic ring and that no carbons adjacent to the
carbons of the double bond are carbonyl.
Representative olefins are:
Olefin Rl R2 R3 . R4
__ _
ethylene H H H H
propylene CH3 ~ H H
5 butene-l C2H5 H H
pentene-1 C3H7 H 11 H
octene~l C6H13 H H H
decene-l C8H17 H H H
.dodecene-l CloH21 H H H
10 isobutylene CH3 CH3 H H
cis-butene-2 CH H CH H
3 3
trans-butene-2 CH3 H H CH3
2-methyl-butene-2 CH3 CH3 CH3 H
1,3-butadiene CH2=CH H H H
lS 1,4-pentadiene H2C=CHCH2 H H H
isoprene H2C=C(CH3) H H H
1,7-octadiene H2C=cH(cH2)4 H H
The broad definition of open-chain olefins
includes olefins where Rl, R , R3, and/or R can be an
aromatic or heteroatom-containing group, provided that
the substituents are inert to the prescribed reaction
condition, or do not deactivate t:he normally reactive
carbon to carbon double bond susc:eptible to halohydrin
formation and further provided that the conditions pre-
viously set forth for the selection of Rl, R , R andR are observed,
Representative olefins containing such aro-
matic or heteroatom groups are:
Olefin Rl R2 R3 R
styrene C6H5 H H H
allyl alcohol HOCH2 H H H
allyl chloride ClCH2 H H H
allyl bromide BrCH2 H H H
The broad definition of olefins also includes
olefins containing cumulated double bonds, such as
allene: .
--6--
CH2 -- C = C1~2
The present invention makes use of certain per-
oxidases which have catalytic acti~ity with respect to
breaking the double bond of olefin compounds and promo-
ting hydroxylation on one of the carbons while promotinghalogenation on the adjacent carbon under particular re-
action conditions. The peroxidase enzymes capable of
providing catalytic acti~ity as described herein are
referred to as "halogenating enzymes".
A preferred halogenating enzyme is derived
from the microorganism Caldariomyces fumago. Other
sources of halogenating enzyme include seaweed, milk
(lactoperoxidase), thyroid (thyroid peroxidase), leuko-
cytes (myeloperoxidase) and horseradish (horseradish
peroxidase). Certain of these peroxidases are commerci-
ally available.
For ease of discussion, various aspects of the
present invention will be described with particularity,
but not exclusively, in connection with use o~ the pre-
ferred peroxidase, chloroperoxidase, deri~ed from Cal-
dariomyces ~uma~o. The microorganism, Caldariomyces
, may be grown as a static or agitated, submerged
culture in Czapek-Dox medium at room temperature for
3 to 10 days by conventional methods. The halogenating
enzyme, chloroperoxidase, is prepared from an aqueous
homogenate of the mycelial pads of the microorganism
grown under static conditions or from the filtrate of
the microorganism grown under static or agitated sub-
merged culture conditions.
The halogenating enzyme may also be used in an
immobilized form. Processes for enzyme immobilization
are familiar to those skilled in the art, and include
reacting either a solution of the enzyme or a suspension
of enzyme containing cells with one of a broad range o~
organic and inorganic supports. Included among these
are polyacrylamide, ethylene-maleic acid copolymers,
~ 5~t~
methacrylic-based polymers, polypeptides, styrene-based
polymers, agarose, cellulose, dextran, porous glass
beads, and aluminum or titanium hydroxide. Enzymes in
this form have increased stability, extended life and
usefulness, and recoverability. Reactions employing
immobilized enzymes may be run in columns or reac-tion
tanks.
In addition to the halogenating enzyme, a
source of inorganic halide and an oxidi~ing agent are
required in the reaction mixture. A preferred oxidizing
agent is hydrogen peroxide, which may be added directly
to the mixture in a single batch addition, or in a con-
tinuous slow feed. It may alternatively be generated
as a slow feed in situ by the use of a hydrogen peroxide-
producing enzyme system. Such enzyme systems are wellknown in the art, and include glucose oxidase in the
presence of glucose, D- and L-amino acid oxidases in
the presence of D- and L-methionine, methanol oxidase
in the presence of methanol, and diamine oxidases in
the presence of histamine. The hydrogen peroxide-genera-
ting system may be present in the non-immobilized or im-
mobilized state as with the halogenating enzyme. The
hydrogen peroxide may also be generated by a chemical
reaction, such as the anthraquinone or isopropyl alcohol
oxidation processes.
With in situ generation of hydrogen peroxide
using glucose oxidase or methanol oxidase, coproducts
include gluconic acid (in the case of glucose oxidase)
and formaldehyde (in the case of methanol oxidase).
Although each of these coproducts is commercially use-
ful, it is conceivable that if the method of the inven-
tion is adopted on a large scale for the production of
epoxides and glycols, the amount of coproduct produced
by in situ hydrogen peroxide generation could exceed
market demand by a substantial amount. Under such cir-
cumstances, two possibilities are presented~ The first
6~
is to find additional uses and therefore additional
markets for the coprod~lcts gluconic acid or formalde-
hyde. The other possibility, however, is to develop
or modify the process in such a way as to produce a
coproduct which is capable of being absorbed by rela-
tively higher market demandO
It may therefore be preferable that the en-
zyme used for the production of hydrogen peroxide
~ n situ be glucose-2-oxidase. Using glucose as a sub
strate, glucose-2-oxidase catalyzes the following re-
action (Volc et al, Folia Microbiol. 23:292-298, 1978):
CHO CHO
H-C-OH C=O
HO C-H HO-C-H
¦ 2 ~ I ~H22
H-C-OH H-l_OH
H-C-OH H-CI~OH
CH2H CH20~I
20 D-glucose D-arabino~2-hexosulose
The D-arabino-2-hexosulose thus produced may be readily
converted to D-fructose by simple chemical hydrogena-
tion as follows:
~HO CH2OH
C=O C=O
HO-C-H catalyst ¢
H~C-OH H C-OH
H-C-OH H-C-OH
CH2 CH2H
D-arabino-2-hexosulose D-fructose
The advantage of relatively low-cost coproduc-
tion of D-fructose is the high desirability of fructose
as a sweetener. The substantially higher sweetness of
fxuctose per calorie or unit weight as compared with
dextrose or sucrose offers distinct advantages for a
wide variety of focd applications. The current price
and restricted availability of fructose, however, has
limited its use. The present invention would be capable
of generating large quantities of fructose at prices
competitive ~7ith sucrose as currently provided.
The hydrogen peroxide is present preferably
in a molar ratio of from about 0~5:1 to about 5~
most preferably in a ratio of about 1:1 or less with
respect to the olefin. The molar ratio preferences
refer to the average presence of hydrogen peroxide
during the reaction. The actual molar ratio will usu-
ally vary during the reaction and the molar ratio at
any particular time may be above or below the ranges
cited. Other suitable oxidizing agents include organic
pero~ides, such as methyl, ethyl, or butyl peroxides.
The halogen source may be any of the water-
soluble halide salts. The preferred halogen sources
are the chlori~e, bromide, and iodide salts of the alka-
li metals, sodium and potassium. The salts are present
in the reaction mixture at a level sufficient to provide
a slight excess of halide ion with respect to the sto-
ichiometric amount required for the reaction.
The reaction is conducted within the pH range
of from about 2.2 t~ about 8Ø The pH of the reaction
may be maintained within the desired range by use of a
bufferin~ agent. Suitable buffers include sodium or
potassium phosphate, gluconate, citrate, formate, and
acetate based systems. Other suitable techniques be-
sides buffering may be used for pH control and adjust-
ment. The reaction is preferably conducted in an aque-
ous medium. While some of the olefins that can be con-
verted by the process are substantially insoluble in an
aqueous medium, the reaction, nevertheless, occurs sati~
factorily under conditions of mixing, or other modes of
-- dispersion, which provide sufficient substrate solubility
for the reaction.
It is also contemplated that the reaction can
1~-
be conducted in the presence of low levels of organic
solvents, such as lower aliphatic alcohols, ketones, di-
oxane, or dimethylformamide to increase substrate solu-
bility. The reaction is preferably conducted under
aerobic conditions and in the temperature range of 15
to about 50~, preferably about 20 to about 30.
A resulting intermediate product in the method
of the present invention is a halohydrin represented
by the following structure:
OH X
C - C - X = halogen
- The halogen is predominantly attached to the
carbon atom that yields the least stable carbonium ion.
Thus, from monoolefinic hydrocarbons such as l-olefins,
there is obtained l-halo-2-hydroxy hydrocarbons tmajor)
and 2-halo-1-hydroxy hydrocarbons (minor):
OH X X OH
R CH CH2 CH2 + R - CH - CH2
(major) (minor)
From polyolefinic hydrocarbons, there may be
obtained both monohalohydrins and polyhalohydrins, as
in the case of 1,3-butadiene:
0~, X
CH2 = CH - ~H = CH2i,CH2 = CH ~ CH - CH2
~H,X OH,X
From olefins containing cumulative double
bonds, there may be obtained both monohalohydrins and
polyhalohydrins, as in the case of allene:
CH2 = C = CH2 ~ CH2 = C - CH2~ CH2 -- 1 -- C~2
OH X
The components other than the olefin~ namely
the halogenating enzyme, the oxidizing agent, the halide
6~
ion soux~e and the bu~fer agent, are simply mixed to-
gether in w~ter or mixed aqueous and organic media to
provide a reaction mi~ture.
The halohydrins formed by the reaction are
easily converted to the oxide, which may itself be use-
ful or which may be converted to a glycol or other use
ful derivative of the olefin in accordance with conven-
tional procedures. To convert the halohydrin to an
epoxide, any of several techni~ues may be employed.
For example, the halohydrin may be converted to the
epoxide by contacting with an aqueous slurry of slaked
lime. Hydration of ~he epoxide results in the glycol.
In a preferred embodiment of the present in-
vention~ the conversion of the halohydrin to an epoxide
is effected by means of an epoxidase used either in a
free or immobilized form. One such enzyme is the halo-
hydrin epoxidase of Flavobacterium spO cells. Similar
activity has been detected in a variety o~ other or-
ganisms such as the fungus Caldariomyces fumago ~nd the
seaweed Laurencia pacifica. The enzyme carries out the
.
~ollowing reaction:
enzyme
halohydrin - ~ epoxide
Specifically for propylene bromohydrin:
CH3 - CHOH - CH2Br or CH3 - CHBr - C~20H~ CH3 Cl~ - C~2
.
For this embodiment, the steps of the method
of the invention for the production of an epoxide from
an alkene may be carried out sequentially. In a most
preferred embodiment the steps are carried out simul-
taneously in the same reactor and the alkene is conver-
ted directly to the epoxide solely through use of en-
zymes without recovery or isolation of a halohydrin in-
termediate.
As previously indicated, the components of the
reaction mixture, namely the olefin, the halogenating
-12~
enzyme, the oxidi~ing agent, the halide ion source, and
the huffering agent are simply mixed together in water
or mixed aqueous or organic media, and agitated for a
period of from about 30 seconds to about 1 hour to ob-
tain the halohydrin. Lower linear olefins, such as ethy-
lene or propylene, which are gaseous, can be reacted
upon by simply passing the gaseous olefins through the
reaction mixture. Equally important and of commercial
significance is the discovery that (1) the same equip--
ment can be used regardless of the specific nature ofthe al~ene, and (2) mixed alkene substrates can be si-
multaneously converted to their respective halohydrins.
Mixtures of alkenes can provide increased total solu-
bility of the alkenes in water (and thus increased con-
version rate) and can improve productivity of the enzymesystem. Of commercial significance also is the ability
to use crude substrates or substrate mixtures, thus
reducing the cost of raw materials. The intentional in-
clusion of small controlled amounts of a given alkene
into the lar~er stream of another enables the economic
production of a low-volume specialty product. Thus,
for example, the halohydrins or oxygenated derivatives
of butadiene could be co-produced with propylene halo-
hydrin, epoxide or glycol in a propylene predominant
system.
The following examples illustrate various
features of the invention, but are in no way intended
to limit the scope of the invention which is defined in
the appended claims. In many of the examples, prepara-
tion of only the intermediate halohydrins is discussed,
since conversion of the halohydrin to the epoxide can
be achieved in all cases by suitable procedures such as
alkaline or enzymatic treatment.
E~amples 1-18
Gaseous Olefins
~____
Dilute hydrogen peroxide (1 mg/ml final),
~6~3
-13-
halide salt (10 mg/ml final) and O.lM potassium phos-
phate buffer are mixed together in a 100 milliliter Py-
rex flask at room temperature and room pressure. The
halogenating enzyme is added and the gaseous olefin is
bubbled into the reac-tion mixture. Sample is withdrawn
at 30 minutes and analyzed for halohydrin.
The halogenating enzymes are prepared as fol-
lows:
Chloroperoxidase (CP). Mycelial pads of Cal-
dariomyces fumago (ATCC 16373) are grown on potato agar
slants as follows: Sliced potato (200 g) is cooked in
distilled water (500 ml) for 40 minutes and then strained.
A solution of glucose (21 g) and agar (20 g) in dis-
tilled water (500 ml) is added to the strained solution.
The pH iS adjusted to 6.8 and the volume is brought to
1 liter with distilled water. The medium is sterilized
at 121 for 15 minutes. The organism is inoculated on
the potato agar slants, produced in accordance with the
above procedure, and is grown for about one week at
room temperature. The organism :is then used to inocu-
late the soybean-glucose medium (50 ml) prepared as fol-
lows: to 1 liter of distilled water are added extrac-
tion process soybean meal (30 g) r glucose (30 g), and
CaCO3 (7 g). The medium is ster:ilized at 121 for 30
~5 minutes and is then inoculated w:ith the organism after
cooling. The organism is grown for 4-5 days on a ro-
tary shaker at 25. 5 ml of this material is used to
inoculate a 500 ml Erlenmeyer flask containing 100 ml
of a modified Czepek-Dox medium prepared by adding the
following to 1 liter of distilled water: NaNO3 (3 g),
KH2PO4 (1 g)l KCl (0.5 g), M~SO4.7H2O tlO mg) and glu-
cose t40 g). The medium is sterilized at 121 for 20
minutes prior to inoculation with the organism. The
organism is grown under static conditions at room tem-
perature 5-7 days~ The black mycelial pads which form
are collected, rinsed with distilled water, and stored
65~
-14-
in plastic bags in a freezer at -10 for subsequent use~
The halogenatinq enzyme is prepared by yrind-
ing 6 mycelial pads (prepared in accordance with the
above procedures) with 60 g acid-washed sand and 60 ml
distilled water for 2 minutes in a Vir-tis 45 homogenizer.
The homogenate is centrifuged while cold and the super-
natant solution filtered through Whatman #l paper at
room temperature. The filtrate is concentrated about 10-
fold using a rotary film evaporator at reduced pressure
and temperature less than 35. The concentrate is
chilled at 0 in an ice bath, and prechilled (0)
ethanol is added until 45% ethanol (v/v) is reached.
The mixture is stirred vigorously for 15 minutes, and
then centrifuged at -10 (at 15,000 g) with a 55-34
rotor in a Sor~al RC-5 Superspeed for 15 minutes. The
black sediment is discarded. To the centrifugate,
cooled at 0, is added additional prechilled ethanol
to give 65~ ethanol (v/v). The mixture is slowly stir-
red for 30 minutes at 0, and then centrifuged as be~ore.
The centrifugate is discarded and the precipitate con-
taining the chloroperoxidase activity is dissolvea in a
minimum volume of 0.05M potassiu~ buffer (pH 7). The
enzyme solution is stored at -20. The activity is
measured as 80 monochlorodimedon units/ml. (Ref: Morris,
25 D.R. and Hager, L~P., J. siOl _~hem. 241, 1763 tl966)).
Lactoperoxidase (LP)~ Purchased from Sigma
Chemical Company (Catalogue #L-7129).
Seaweed Perox ase1 (SWPl). ~aurencia pacifi-
ca obtained along the coast of La Jolla, California, is
prepared by grinding and dispersing 5 g of the seaweed
in 10 ml of 0O3M potassium phosphate buffer (pH 6.0) for
2 minutes in a Virtis 45 homogenizer.
Seaweed Peroxidase2 (SWP2). Coralina sp. ob-
tained along the coast of La Jolla, California is ~round
in a Virtis 45 homogenizer for 5 minutes in distilled
water. The homoqenate is spun at 20,000 rpm ~or 20
5~
-15-
minutes~ The supernatant isdecanted and saved~ The
pellet is resuspended in distilled water and recentri-
fu~ed. This supernatant and previous supernatant are
combined~ The solution is brought first to 33%, then
to 55~ saturation in ammonium sulfate. Centrifugation
and separation of pellet is performed at each step.
The 33%-55% pellet fraction is passed through a DEAE
column usins a 0.3M to lM phosphate buffer (pH 6.0)
gradient, The fraction which elutes at lM is dialyzed
against 20 mM phosphate buffer (pH 6) overnight. This
preparation is stored at -20 until needed. The ac-
tivity is measured as 2 monochlorodimedon units/ml.
Horseradish Peroxidase (HRP). Purchased from
Si~ma Chemical Company (Catalogue #P-8250).
The halohydrins are identified by ~as chroma-
tography/rnass spectrometry. The halohydrin and its cor-
respondin~ epoxide (after treatment of the halohydrin
with base) are compared with authentic samples. The
analysis of propylene bromohydrin illustrates the pro-
cedure:
10 ~ of the reaction mixture was injected
into a Perkin Elmer ~odel 3920 gas chromatograph,
equipped with a 6 foot by 1/8 inch coiled, stainless
~teel column, packed with Porapa~ R (80/100 mesh) and
attached to a Dupont Model ~1-491 mass spectrometer
operating at 70 eV ionization. Flow rate was set at
30 ml~minute for helium and the column temperature was
pro~rammed for 185C to 220C at a rate of 1C/minute.
Retention times for the propylene bromohydrins were 9
minutes for 1-bromo-2-propanol and 10 minutes for
2-bromo-1-propanol.
Product identity was confirmed by comparison
with authentic samples of propylene bromohydrin: l-bro-
mo-2 propanol was purchased from Pfaltz and Bauer~ Inc.;
2-bromo-1-propanol was synthesized by lithium aluminum
hydride reduction of l-bromopropionyl chloride. The
~L6~i~3~
-16-
reaction products and the authentic samples showed the
same retention times and identical mass spectra bromine
was identified by the presence of the M and M+2 isotope
clusters of e~lal intensity; the molecular ion for both
isomers was confirmed by chemical ionization with iso-
butane reagent gas (M+; m/e 138~1~0); for 1-bromo-2-
propanol the major ~ragmentation was the expected loss
of CH2Br while for 2-bromo-1-propanol the major frag-
mentation was the expected loss of CH3CHBr.
Further, both bromohydrins were converted to
propylene epoxide by addition of 10~ sodium hydroxide
to their aqueous solutions to yield a pH greater than
10. Identity of propylene epoxide was confirmed by gas
chromatography/mass spectrometry comparison with an
authentic sample (purchased from Aldrich Chemical Com-
pany, Inc.). The reaction product after base treatment
and the authentic sample showed the same retention time
(2 minutes) and identical mass spectxa (M~î m/e 58).
Variable conditions and results are set forth
in Table I.
The reactions are found clean, i.e., no de-
tected halogenated byproduct formation.
~ - ~
~I co u~ ~ o ~ o Lt) ~ ~ u~
~ o ~ o o o ~ o ~i o o r'i o o o o o o ~`~
k _ ~
o o
,~ ,~
~ ' ~ ~ ~ t r~
a~ - - o o
-- -- o o o o o ~
,~ O 0~ T
o~
~, ~, o~ ~o ~o ~o
o o ~ ~ ~ ~ ~ao o o .~ ,~
o + ~ c`~ l o~ ~
~ ~ + -~ ~ + ~ '~ 7 ~
H ~ h ~ ~ ~ b . ~
~ o~ ~ ~ o~ o o~ o~ o ~ ~ o~ ~ o~ o~ o~ o~ o~ ~ ~
.
o ~ v~l
.
r~ r~ r~ ~I r~ r~ ~I r~ ~1
Examples 19-31
Liquid or Solid Olefins.
Dilute hydro~en peroxide (1 mg/ml final), po-
tassium bromide (10 mg/ml ~inal), liquid or solid ole-
fin (5 mg/ml final) and 0.1M potassium phosphate buffer
~pH 3) are mixed together in a 100 milliliter Pyrex
flask at room temperature and room pressure. Chloro- ~
peroxidase (10 units; prepared as in Examples 1-18~ is
added. Sample is withdrawn at 30 minutes and analyz~d
for halohydrin.
The halohydrins are identified by gas chroma-
tography/mass spectroscopy. The halohydrin and its cor-
responding epoxide (after treatment of the halohydrin
with base) are compared with authentic samples. The
analysis of allyl bromide bromohydrin (Example 27) il-
lustrates the procedure.
10 ~1 of the reaction mixture was injected
into a Perkin Elmer Model 3920 gas chromatograph,
equipped with a 6 foot by 1/8 inch coiled, stainless
steel column, packed with 3~ OV225 on GAS-CHROM Q
(100/120 mesh), and attached to a Dupont Model 21-491
mass spectrometer operating at 70 eV ionization. Flow
rate was set at 30 ml/minute for helium and the column
temperature was programnted from 140~ to 200~ at a rate
o~ 2C/minute. Retention times for the allyl bromi~e
bromohydrins were 17 minutes for 1,3-dibromo-2-propanol
and 20 minutes for 2,3-dibromo-1-propanol.
Product identity was confirmed by comparison
with authentic samples of the bromohydrins. (Both were
purchased ~rom Aldrich Chemical Company, Inc.). The
reaction products and the authentic samples showed the
same retention times and identical mass spectra: 2 bro-
mines per molecule were identified by the presence of
the M, M+2 and M+4 isotope clusters of 1:2:1 in~ensity;
the molecular ion for both isomers was visible (M~; m/e
216+218+220); for 1,3-dibromo-2-propanol the major
5~
-19-
fragmentation was the expected loss of CH2Br while for
2,3-di.bromo-1-propanol the major fragmentation was the
expected loss of CH2BrCH2Br.
Further, both bromohydrins were converted to
epibromohydrin (i.e. the epoxide of allyl bromide bromo~
hydrin) by addition of 10~ sodium hydroxide to their
aqueous solutions to yield a p.H> 10. Identity of epi-
bromohydrin was confirmed by gas chromatography/mass
spectrometry comparison with an authentic sample (pur-
chased from Aldrich Chemical Company, Inc.). The re-
action product after base treatment and the authentic
sample showed the same retention time (5 minutes) and
identical mass spectra (M ; m/e 136~138).
Results are set forth in Table II.
1~16S43
Y ~ N
b h b b ~ ~ ~ a.
~ ~ ~I ~
!~ N N N ~I N N N N ~i N N r I ~
~ ~ b
1~ ~ N~ N
I S~ N t~ ~ LJ
~ Y~ b b ' ~ ~
O <''I N N ~ N ~ N ~ p N ~ t-- J~
o ~ ~ ~ ~
~ O 1--l N ~ ~ O r-l
1~ ¦ ~1 ~ N ~1 N N N N N N N ~ ~7
i5~
-21--
E_ ple 32. The procedure of Examples 2 and 6
are followed substituting a mixed ethylenepropylene
stream or the single olefin.
The result is 1.4 mg/ml of 2-bromoethanol and
0.9 mg/ml of 1-bromo-2-pxopanol + 2-bromo-1-propanol
(90~
Example 33. The importance of immobilizing
the halogenating enæyme and using a slow feed of H2O2
by ln situ enzymatic generation are shown in this ex-
ample. Halide salt (10 mg/ml final) and O.lM potassiumphosphate buffer (pH 6) are mixed together in four 100
milliliter Pyrex flasks at room temperature and room
pressure. Propylene is bubbled into the reaction mix-
tures. Sample is withdrawn at 60 minutes and analyzed
for halohydrinO The variable conditions and the results
are set forth in Table III. These results indicate that
the use of immobilized halo~enating enzyme coupled with
a slow feed (slow to the point oE use as generated) of
H2O2 greatly improves the production of propylene bromo-
hydrins, from which propylene oxide is easily obtained.
TABLE III
Propylene
Bromohydrin
~eaction H2O2Feed Lactoperoxidase Yield mg/ml
1 Direct addition (1) N~n-in~.obilized (3) .008
2 Direct addition (1) ~m~bilized (4) .03
3 In situ generation~)Non-in~obilized (3) .21
4 In sit~lgeneration~)Immobilized (4) 1.51
Footnotes: -
(1) 1 mg/ml H202 final
(2) 0.2 ml of l.OM a-D-glucose and 0.1 ml of glucose oxidase
SSi~ Ch~cal Cbrpa~ Catalogue ~6500) per 10 ml of re-
action mLxture.
(3) from P.L. Biorhemicals~ ~lC., 20 units added.
(4) from P.L. Biochemicals, ~c., bound to Sepharose, 20 units
added.
-22-
Example 34. The use of immobili~ed seaweed
peroxidase is shown in this example.
The immobilized seaweed peroxidase is pre-
pared as follows:
Glass beads (obtained from Sigma Chemical
Company, PG-700-200) are activated by suspending 1 g
of glass beads in 18 ml of deionized water. 2 ml of
10~ (v/v ~-aminopropyltriethyoxyl silane are added and
the pH oE the mixture is adjusted to 3-5 with 6N HCl.
The mixture is shaken at 75C for 2 hours. The glass
beads are then vacuum dried overnight at 80C. 3.2 ml
of purified Coralina sp. enzyme, prepared as in Ex-
amples 1-18, and 50 mg. of water soluble carbodiimide
are added to the glass beads. The pH is adjusted to
4.5, and the mixture is then shaken at 4C overnight.
The product - enzyme coated beads - is washed with
water. The activity is measured as 2 monochloroclimedon
units/g of beads.
A reaction using 1 g of the seaweed peroxidase
coated glass beads is run as in Example 33, reaction 4,
with these modifications.
a) 40 mg/ml KBr final
b~ 50 mg/ml ~,D-glucose
c) 1.0 ml of glucose oxidase
25The result is:
Reaction time, Propylene Bromohydrin
hour _ (mg/ml?
-
4 9.0
8 13.3
30 10 16.S
21 24.1
Example 35. The procedure of Example 34 is
followed substituting KI for KBr.
The result is 3.0 mg/ml of propylene iodohy-
drin at 1 hour.
Example 36. The use of cells of Hansenula
5~3
-23-
pol~morpha ~TCC 260]2 as a source of methanol oxidase
to generate I-12O2 is shown in this example.
Cells of the microorganism are prepared as
follows:
The culture is maintained on agar slants at
37, with periodic transfer. The composition of the
agar medium per liter is NaNO3 (3 g), KCl (0~5 g),
MgSO4.7~2O ~0.5 g), acidified 1% FeSO4.~H2O (1 ml),
~KI~2PO4 ~1 g), glucose (40 g) t NaCl (32.14 g), yeast
extract (2 g), and agar (15 g). pH is adjusted to
6.0 with 10% NaOE~ prior to sterilization under standard
conditions. Growth of the culture to be used as a
source of methanol oxidase activity is carried out on a
medium of the following composition per liter:
(NH4)2 HPO4 (6 g), MgSO4.7H2O (2 g~, and yeast extract
(5 g). pH is adjusted to 5.0 with 85% H3PO4 prior to
sterilization under standard conditions. Fermentations
are run in 125 ml Erlenmeyer flasks containing 20 ml of
this medium to which 0.1 ml sterile methanol is added.
20 The fermentations are performed at 37, 200 rpm on a
New Brunswick sha]~er, 2"-throw, for 1-3 days. The cells
are harvested by centrifugation, washed once with 0.lM
phosphate buffer and then resuspended at 1/5 the origi-
nal fermentation volume in 0.1M phosphate buffer, pH
6Ø The cells are stored in the refrigerator at 4C
for up to one week without substantial loss of activity.
A reaction with lactoperoxidase is run as in
Example 33, reaction 3 with these modifications:
a) 0~1 ml 3% methanol instead of 0.1 ml lM
a-D-glucose.
b) 1 ml of above-described suspension of
cells of Hansenula polymorpha ATCC instead of 0.1 ml
glucose oxidase.
The reaction mixture is analyzed for propy-
lene bromohydrins after 60 minutes~ The total ~ieldis 175 ~g/ml.
5'~3
-24-
Example 37. The ability to convert propylene
_
to propylene epoxide, enzymatically, without isolation
of the intermediate halohydrin is shown in this Example.
Flavobacterium sp. (Cetus #5095) contains a halohydrin
5 epoxidase, which converts halohydrins to epoxides.
The microorganism is grown at 25, 200~pm 2"-
throw on a New Brunswick shaker in a medium reported in
the literature (C. E. Castro and E. Wu sartnicki~ Bio-
chemistry 7:3213 (1968)). 100 ml of medium is used per
500 ml Erlenmeyer flask~ The microorganism is main-
tained on slants using an agarized version of the liquid
medium. The seed stage is usually 48 hours. The cells
~or use in the conversion reaction are then grown for
an additional 2-3 daysr using a 1-5% inoculum from the
seed stage.
A washed cell preparation of the microorganism
is prepared ~y standard procedures and is finally resus-
pended at a twenty-fold concentration (as compared to
the concentration in the growth stage flasks) in the
appropriate phosphate buffer. Dry weight of cells in
these washed cell suspensions is 15 mg/ml.
~ he procedure o~ Example 8 is followed, with
the addition of 30 mg of Flavobacterium sp. cells to
the reaction mixture.
2~ The results obtained are 0.2 mg/ml propylene
bromohydrin and 0.3 mg/ml propylene epoxide.
Example 38. The procedure of E~ample 37 is
~ollowed except ethylene is substituted for propylene
and chloroperoxidase ~2 units~ is substituted for lacto-
peroxidase.
The results obtained are 100 ~g/ml ethylenebromohydrin and 5 ~/ml ethylene epoxide.
Example 39. The ability to produce oxide
from propylene in an integrated immobilized enzyme/cell
system in a continuous-flow column configuration is
flemonstrated in this example. The ~eneral conversion
proceeds in the following manner:
Immoblized Br OH
glucose oxidase CH2-CH-CH3
CH2=CH--CH3 > +
immobilized OIH Br
chloroperoxidase CH2-CH-CH3
Propylene Propylene Bromo-
hydrins
Immobilized ~O\
balohydrin CH2-CH-CH3
epoxidase
. ,~
(Flavobacterium
sp. cells)
Propylene
Oxide
By way of example, the glucose oxidase can be
co-valently immobilized with AH-Sepharose 4B; the chloro-
peroxidase can be ionically immobilized on DEAE-sepha-
dex A50 beads, and the halohydrin epoxidase can be in-
solubilized inside the cells with polyacrylamide gel.
By way of example, one column configuration
includes a first region which contains the initial mix-
2S ture (50 ml) of reaction components: propylene gas con-
tinuously bubbled in, 0.42M KBr, 0.01M glucose, and
phosphate buffer (pH 4.4, 0.3M). The mixture is slowly
dripped onto a column of immobilized chloroperoxidase
(CP) and glucose oxidase (GO) prepared by mixing 5 ml
CP-beads and 1 ml GO-beads. This is the first stage of
the system to produce propylene bromohydrin.
The details of immobilizing glucose oxidase
and chloroperoxidase follow:
The glucose oxidase (1460 units/ml) is pur~
chased from Sigma Chemical Company. The insoluble beads
of A~J-Sepharose 4B are obtained from Pharmacia Fine
- 6-
Chemical Company. Both enzyme and beads are adjusted
to pH 5Ø To immobilize the enzyme onto the beads,
10 ml glucose oxidase and lO ml beads are mixed. The
coupling reaction is initiated by the addition of 2 ml
N-cyclohexyl-~'(2-(4-methyl-morpholino)-ethyl)-carbodi-
imide solution (100 mg/2ml). The reaction mix-ture is
incuhated at 4C overnight. The beads are then washed
with 0.03M phosphate buffer (pH 4.4). The glucose oxi-
dase-AII-Sepharose 4s beads are stored at 4C for use.
The chloroperoxidase (prepared as in Examples
1-18) is immobilized by mixing equal volume dialyzed
chloroperoxidase with hydrated DE~E-Sephadex A50 beads.
The enzyme-beads complex are washed three times with
0.03M phosphate buffer (pH 4.4). The activity of the
beads is estimated to be 2.17 units/ml with monochloro-
dimedon assay. The immobilized chloroperoxidase is
stored at 4C for future use.
The resulting eluate, containing excess re-
agents as well as propylene bromohydrin and gluconic
acid, is adjusted to pH 6 with phosphate buffer (pH 6,
l~OM) to allow for substantial activity with halohydrin
epoxidase contained in the next column region. The im-
mobilization of halohydrin epoxidase is performed by
immobilizing the intact cells of Flavobacterium sp.
(prepared as in Example 37)~with the following reaction
mixture:
Flavobacterlum sp. 4 gm (wet weight)
Saline Solution 4 ml
Acrylamide monomer 750 mg
Bis-acrylamide 40 mg
TEMED 25 ~
Ammonium persulfate 0.5 ml (2.5%)
The polymerized gel is then blended into beads
with a Waring ~lender (low speed, 20 seconds)~ This is
the second stage of the system, producing propylene
oxide from the propylene bromohydrins of the firs-t stage.
ii4~
~27-
The results obtained are 75 ~g/ml propylene
bromohydrin and 5 ~g/ml propylene epoxide at a flow
rate through the column of 1 ml/hour.
The foregoing description and accompanying
examples therefore demonstrate that epoxides and glycols
may be produced from alkenes at room temperature. The
process requires essentially no energy additions and is
applicable to a wide variety of substrates including
gaseous alkenes such as ethylene and propylene. Vari-
ous modifications of the invention will become apparentto those skilled in the art from the foregoing descrip-
tion. Such modifications are intended to fall within
the scope of the following claims.
